The goal of this project is to obtain new information about the structure, organization and mechanism of action of the enzyme Na,K-ATPase. This membrane-bound enzyme is responsible for the active transport of Na ion and K ion across cell membranes. It is also the putative receptor site for the cardiac glycosides. This enzyme is of special interest because of its role in the maintenance of the electrophysiological properties of heart muscle cells. I will be continuing with studies of Na,K-ATPase which utilize antibodies raised to the enzyme. My major objective is to isolate for the first time monospecific antibodies to individual antigenic determinants of the enzyme. This is done by fusing antibody-producing mouse spleen cells with a cultured mouse myeloma cell line which makes possible the in vitro production of the spleen cell antibodies. Hybrid cells with the ability to produce anti-Na,K-ATPase antibodies are then identified, isolated and cultured so that their antibodies can be collected. These antibodies will be used to study specific antibody-enzyme interactions, the interactions between the different ligand binding sites of the enzyme, and to possibly locate the in vivo cardiac glycoside receptor, the active site, and ligand binding sites of the enzyme using immunocyto-chemical techniques. In addition, various immunochemical techniques utilizing holoenzyme, catalytic subunit, glycoprotein subunit, and digoxin specific antibodies will be used to determine the influence of the holenzyme conformation on its antigenic properties and to try to identify and isolate peptide fragments of the enzyme which contain the cardiac glycoside receptor, the phosphorylation site and cation binding sites, and to aid sequence work which is already underway.